Difference between revisions of "Team:CCU Taiwan/InterLab"

Background

All of the 2018 iGEM teams are invited and encouraged to participate in the Fifth International InterLaboratory Measurement Study in synthetic biology.” Our team took part in this study which aimed to be one of the labs to make the calibration in different condition. In our case, we measured fluorescence using plate reader.

Interlab Project

OD 600 Reference point - LUDOX Protocol
Particle Standard Curve - Microsphere Protocol
Fluorescence standard curve - Fluorescein Protocol
Cell measurement protocol

Experiment

Calibration 1: OD 600 Reference point - LUDOX Protocol

Materials：

1ml LUDOX CL-X (provided in kit)

ddH₂O (provided by team)

96 well plate, black with clear flat bottom preferred (provided by team)

Method：

Add 100 μl LUDOX into wells A1, B1, C1, D1

Add 100 μl of dd H₂O into wells A2, B2, C2, D2

Measure absorbance at 600 nm of all samples in the measurement mode you plan to use for cell measurements

Record the data in the table below or in your notebook

Import data into Excel sheet provided ( OD600 reference point tab )

Data Result：

Calibration 2: Particle Standard Curve - Microsphere Protocol

Materials：

300 μL Silica beads - Microsphere suspension (provided in kit, 4.7 x 10⁸ microspheres)

ddH₂O (provided by team)

96 well plate, black with clear flat bottom preferred (provided by team)

Method：

Prepare the Microsphere Stock Solution:

Obtain the tube labeled “Silica Beads” from the Interlab test kit and vortex vigorously for 30 seconds.

NOTE: Microspheres should NOT be stored at -20 degrees Celsius or below , as freezing affects the properties of the microspheres. If you believe your microspheres may have been frozen, please contact the iGEM Measurement Committee for a replacement (measurement at igem dot org).

Immediately pipet 96 μL microspheres into a 1.5 mL eppendorf tube

Add 904 μL of ddH₂O to the microspheres

Vortex well.

Data Result：

Calibration 3: Fluorescence standard curve - Fluorescein Protocol

Materials：

❏ Fluorescein (provided in kit)
❏ 10ml 1xPBS pH 7.4-7.6 (phosphate buffered saline; provided by team)
❏ 96 well plate, black with clear flat bottom (provided by team)

Method：

Prepare the fluorescein stock solution:
❏ Spin down fluorescein kit tube to make sure pellet is at the bottom of tube.
❏ Prepare 10x fluorescein stock solution (100 μM) by resuspending fluorescein in 1mL of 1xPBS. [ Note : it is important that the fluorescein is properly dissolved. To check this, after the resuspension you should pipette up and down and examine the solution in the pipette tip – if any particulates are visible in the pipette tip continue to mix the solution until they disappear.]
❏ Dilute the 10x fluorescein stock solution with 1xPBS to make a 1x fluorescein solution with concentration 10 μM: 100 μL of 10x fluorescein stock into 900 μL 1xPBS

Prepare the serial dilutions of fluorescein:
❏ Add 100 μl of PBS into wells A2, B2, C2, D2....A12, B12, C12, D12
❏ Add 200 μl of fluorescein 1x stock solution into A1, B1, C1, D1
❏ Transfer 100 μl of fluorescein stock solution from A1 into A2.
❏ Mix A2 by pipetting up and down 3x and transfer 100 μl into A3…
❏ Mix A3 by pipetting up and down 3x and transfer 100 μl into A4...
❏ Mix A4 by pipetting up and down 3x and transfer 100 μl into A5...
❏ Mix A5 by pipetting up and down 3x and transfer 100 μl into A6...
❏ Mix A6 by pipetting up and down 3x and transfer 100 μl into A7...
❏ Mix A7 by pipetting up and down 3x and transfer 100 μl into A8...
❏ Mix A8 by pipetting up and down 3x and transfer 100 μl into A9...
❏ Mix A9 by pipetting up and down 3x and transfer 100 μl into A10...
❏ Mix A10 by pipetting up and down 3x and transfer 100 μl into A11...
❏ Mix A11 by pipetting up and down 3x and transfer 100 μl into liquid waste TAKE CARE NOT TO CONTINUE SERIAL DILUTION INTO COLUMN 12.
❏ Repeat dilution series for rows B, C, D
❏ Measure fluorescence of all samples in instrument
❏ Record the data in your notebook
❏ Import data into Excel sheet provided ( fluorescein standard curve tab )

Data Result：